EPR detection of sulfanyl radical during sulfhemoglobin formation - Influence of catalase.

Hemoglobin in its ferryl form oxidizes hydrogen sulfide and is transformed to sulfhemoglobin, where the sulfur is inserted covalently at the heme edge. Shown here is evidence that-as previously proposed by others-this process involves oxidation of hydrogen sulfide to a sulfanyl radical detectable by spin-trapping in electron paramagnetic resonance (EPR) spectroscopy. The yields and rates of formation of sulfhemoglobin as well as of the sulfanyl radical are affected by the same factors that affect the reactivity of hemoglobin ferryl, in bovine hemoglobin and in phytoglobins as well. A freely-diffusing sulfanyl radical is thus proposed to be involved in sulfhemoglobin formation. Catalase is shown to accelerate this process due to a previously described hydrogen sulfide oxidase activity, within which EPR evidence for sulfanyl generation is shown here for the first time. The reaction of preformed ferryl with hydrogen sulfide-in absence of hydrogen peroxide-is studied by stopped-flow at several pH values and explained in light of reactivity and redox potential control.

Structural determinants for the formation of sulfhemeprotein complexes.

Several hemoglobins were explored by UV-Vis and resonance Raman spectroscopy to define sulfheme complex formation. Evaluation of these proteins upon the reaction with H(2)O(2) or O(2) in the presence of H(2)S suggest: (a) the formation of the sulfheme derivate requires a HisE7 residue in the heme distal site with an adequate orientation to form an active ternary complex; (b) that the ternary complex intermediate involves the HisE7, the peroxo or ferryl species, and the H(2)S molecule. This moiety precedes and triggers the sulfheme formation.

Chemiluminescence of hemoglobin and identification of related compounds with the hemoglobin chemiluminescence in plasma.

A low level of chemiluminescence by hemoglobin (Hb) was detected in the reaction with H2O2 and hydrogen donors such as gallic acid and catechins. The photon intensity was affected by the ferric state of Hb (methemoglobin > oxyhemoglobin), and was roughly correlated with the radical-scavenging potential of catechins. We hypothesized the reversible activation reaction of Hb as the chemiluminescence mechanism of the H2O2/gallic acid/Hb system. It is indicated that the oxidized-Hb (Hb-OOH) formation was a chemiluminescence-rate-determining step and one-electron reduction by a hydrogen donor of the compound-I-type intermediate ([.XFeIV] = O) proved a chemiluminescence-specificity-determining step. Spectral analysis showed that the photon emission from the H2O2/gallic acid/Hb system was produced without singlet oxygen generation. The concentration dependence of photon intensity suggests a high consumption ratio of H2O2 leading to protection from H2O2 toxicity. Albumin was defined as a hydrogen donor by the isolation of chemiluminescent substance in plasma using this chemiluminescence system.

The incorporation of sulphaem into recombinant adult human haemoglobin produced in a yeast expression system.

The production of adult human haemoglobin in a yeast expression system has been shown to lead to the formation of functional oxygen-binding tetrameric proteins with the incorporation of endogenously synthesized haem. Adachi et al. [(1992) Protein Engng, 5, 807-810] identified two partially resolvable forms of the expressed haemoglobin, one of which showed higher oxygen affinity and lower cooperativity than normal. We show that in contrast to the previously expressed view that the abnormal form is due to abnormal protein folding, that it represents tetrameric haemoglobin containing incorporated sulphaem. Furthermore, the incorporation of sulphaem is shown to be a time-dependent process, with no detectable sulphaem being incorporated prior to 16 h post-induction. Numerical simulation based on our analysis of sulphaem composition gives an excellent fit to oxygen binding data previously reported for samples containing mixtures of normal haemoglobin and sulphaemoglobin.

1H nuclear magnetic resonance study of the prosthetic group in sulfhemoglobin.

The molecular and electronic structure of the modified prosthetic group of sulfhemoglobin (SHb) was investigated by 1H NMR for the low-spin ferric cyano-met and high-spin ferrous deoxy sulfhemoglobin complex. The 1H NMR resonances of the two subunits in the cyano-met SHb complex were differentiated on the basis of the differential stability toward regeneration of native subunits. The subunit origin for the two sets of resonances was established by formation of the sulfglobin protein for the isolated alpha-chain prior to assembling with the native beta-subunit to yield a tetramer with sulfhemin in the alpha-subunits. The subunit peak assignments establish that it is the beta-subunit of SHb which regenerates more rapidly to native protein. The hyperfine shifted sulfhemin peaks were assigned based on steady-state nuclear Overhauser effects which demonstrated that similarly hyperfine shifted peaks exhibit the same dipolar connectivities observed in the analogous sulfmyoglobin complex. Hence it is concluded that pyrrole B is the site of reaction in both hemoglobin and myoglobin. The initially formed SHb complex failed to equilibrate to yield a complex with a sulfhemin sufficiently stable to extraction as found previously for sulfmyoglobin. However, apoHb readily bound the green sulfhemin extracted from the terminal alkaline equilibration product of sulfmyoglobin. The inhibition on the equilibration to the alkaline form with the exocyclic thiolene ring is attributed to the interaction with Val FG5. The observations of the same dipolar connectivities among similarly hyperfine shifted peaks in the directly prepared and reconstituted SHb complexes further support the same structure for the sulfhemin in sulfmyoglobin and SHb. The strongly hyperfine shifted peaks in the deoxy form of both SHb complexes were found very similar to those of the analogous sulfmyoglobin complexes. The proximal His labile ring proton signal appears to experience a 5- to 10-ppm decrease upon conversion of a native globin to sulfglobin. This attenuation may provide a probe for differentiating chlorins and hemins in globin pockets.